Download Progressive Loss of CD8 + T Cell

P r o g r e s s i v e Loss o f C D 8 + T C e l l - m e d i a t e d C o n t r o l o f a
~ / - H e r p e s v i r u s in t h e A b s e n c e o f C D 4 + T Cells
By 1Khonda D. Cardin,James W. Brooks, Sally 1K. Sarawar,
and Peter C. Doherty
From the Department of Immunology, &.Jude Children's Research Hospital, Memphis, Tennessee
38105
Summary
A unique experimental model has been developed for dissecting the integrity of CD8 + T cellmediated immunity to a persistent gammaherpesvirus under conditions of CD4 + T cell deficiency. Respiratory challenge of major histocompatibility complex class II - / and + / +
C57BL/6J mice with the murine gammaherpesvirus 68 (MHV-68) leads to productive infection of both lung and adrenal epithelial cells. Virus titers peak within 5-10 d, and are no longer
detected after day 15. Persistent, latent infection is established concurrently in splenic and
lymph node B cells, with higher numbers of MHV-68 § lymphocytes being found in all lymphoid sites analyzed from the + / + mice concurrent with the massive, but transient splenomegaly that occurred only in this group. From day 17, however, the numbers of infected B lymphocytes were consistently higher in the - / - group, while the frequency of this population
diminished progressively in the + / + controls. Infectious MHV-68 was again detected in the
respiratory tract and the adrenals of the - / - (but not the + / + ) mice from day 22 after infection. The titers in these sites rose progressively, with the majority of the - / - mice dying between days 120 and 133. Even so, some CD8 + effectors were still functioning as late as 100 d
after infection. Depletion of CD8 + T cells at this stage led to higher virus titers in the - / lung, and to the development of wasting in some of the - / - mice. Elimination of the CD8 +
T cells from the + / + group (day 80) increased the numbers o f M H V - 6 8 + cells in the spleen,
but did not reactivate the infection in the respiratory tract. The results are consistent with the
interpretation that CD8 + T cell-mediated control of this persistent gammaherpesvirus is progressively lost in the absence of the CD4 + T cell subset. This parallels what may be happening
in AIDS patients who develop Kaposi's sarcoma and various Epstein Barr virus-associated disease processes.
p
ersistent infection of human B lymphocytes with the
prototypic type 1 y-herpesvirus (yHV) 1, Epstein-Barr
virus (EBV), is thought to be controlled by virus-specific
CD8 + T cells (1-3). Most people are EBV carriers, and
subsequent to the infectious mononucleosis phase that often follows the initial invasion of the virus, normally remain asymptomatic into advanced age, This situation can
change dramatically in the immunosuppressed host with,
for example, EBV-associated lymphoproliferative disease
being a too common complication of therapeutic bone
marrow and organ transplantation (4-6). The progression
of human immunodeficiency virus (HIV) infection to the
TAbbreviations used in this paper: BAL, bronchoalveolarlavage; BM, bone
marrow; CLN, cervical lymph node; CMC, carboxymethyt cellulose;
CMV, cytomegalovirus;EBV, Epstein-Barr virus; 3'HV, 3,-herpesvirus;
i.n., intranasal; LCMV, lymphocytic choriomeningitis virus; MHV-68,
mouse ~HV-68; MLN, mediastinaI lymph node; OMK, owl monkey
kidney (cells).
863
AIDS can also be accompanied by the development of
EBV-associated lymphoma, and by the increased excretion
of EBV from the characteristic oral hairy leukoplakia (7, 8).
Furthermore, individuals with AIDS show a greatly increased incidence (9) of Kaposi's sarcoma. Recent analysis
has implicated a novel type 2 y H V in the etiology of KS
(10, 11). The development of AIDS is associated with declining numbers of CD4 + T cells, the primary target of
HIV replication (12), rather than the loss of the CD8 + set
that is generally considered to be the principal mediator of
immune surveillance against viruses (13).
Experimental dissection of the interaction between a
y H V and the CD4 + and CD8 + T cell responses has been
made possible by the recent development of the murine
~/HV (MHV-68) model (17). Like EBV, this type 2 y H V
(15, 16) is disseminated as a consequence of the productive
infection of mucosal epithelial cells (17). The viremia that
occurs during the acute phase of the infection also leads to
transient virus replication in the thymus and adrenal epithe-
J. Exp. Med. 9 The Rockefeller University Press 9 0022-1007/96/09/863/09 $2.00
Volume 184 September 1996 863-871
lium, and to the establishment of latent infection in large
numbers of B lymphocytes (18). Selective elimination of
the responding C D 4 + or CD8 + T cells in BALB/c (H-2 d)
mice by in vivo treatment with mAbs has established that
the initial stage o f virus replication in epithelial sites is c o n trolled by C D 8 + effectors acting in the absence of the
C D 4 + subset (19). The characteristic splenomegaly (19, 20)
was, however, greatly diminished in the C D 4 + T celldepleted mice, and fewer B cells could be shown to carry
the virus.
The present study extends this analysis to long-term
M H V - 6 8 infection of H - 2 b mice that are constitutively
CD4-deficient as a consequence o f h o m o z y g o u s disruption
(-/-)
of the H 2IA b class II M H C glycoprotein (21).
T h o u g h there are low numbers of C D 4 + T ceils in these
M H C class II - /
animals (2-5% in the periphery), studies with a variety of experimental systems have given no indication that these lymphocytes can function to provide
cognate help for antibody production or to promote CD8 +
T cell responses (21-24).
Materials and M e t h o d s
Mice. The + / + C57BL/6J (B6) mice were purchased from
Jackson ImmunoResearch Laboratories, Inc. (Bar Harbor ME).
The C2D mice that are functionally - / - for the H-IAb gene
(21) were bred under licence from GenPharm International
(Mountain View, CA), at St. Jude Children's Research Hospital.
Female mice were infected with MHV-68 at 8-10 wk of age,
then maintained under otherwise specific pathogen-free conditions in BL3-1evel containment.
Virus Stocks and Plaque Assay. The original stock of MHV-68
(clone G2.4) was obtained from Dr. A.A. Nash at the University
of Cambridge (Cambridge, UK). The virus was grown in owl
monkey kidney (OMK; 1566CRL, American Type Culture Collection [ATCC], 1Kockville,MD) cells maintained in 1KPMI 1640
(GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS,
2 mM L-glutamine, and gentamicin. The OMK cells were infected at a multiplicity of infection (MOI) of 0.01, and virus
stocks were prepared by sonicating the cells in PBS+0.1% BSA
on ice, then stored at -70~
Uninfected control stocks were
prepared from OMK cells in the same manner. Virus titers were
determined by plaque assay on NIH 3T3 cells (ATCC
CIKL1658) grown in DMEM (GIBCO BRL) supplemented with
10% FCS, 2 mM 1-glutamine, 4 mM Hepes, and gentamicin. Dilutions of stock virus, sonicated mouse tissues, or sonicates of
lymphocytes were adsorbed onto the NIH 3T3 monolayers for 1 h
at 37~ then washed with media and overlaid with 2.0 ml ofc0rboxymethyl cellulose (CMC) diluted 1:1 in 2• modified Eagle's
medium supplemented with 10% FCS, glutamine, gentamicin,
and nonessential amino acids (13). After 5 d, the CMC overlay was
removed, and the monolayers fixed with methanol and stained
with Giemsa to determine the number of plaques.
Injection and Sampling. The mice were anesthetized (25) with
Avertin (2,2,2 tribromoethanot), then infected intranasally (i.n.)
with 4 x 1.03PFU of MHV-68 in 40 kcl ofPBS + 0.1% BSA. At
various times after infection, the mice were anesthetized again
and bled from the axilla. The inflammatory cells from the lung
were obtained (25) by bronchoalveolar lavage (BAL), various tissues were taken for virus isolation, and single-cell preparations
were made from the lymph nodes and spleen for cytokine, cyto864
toxicity, flow cytometry, and infectious center assays. Peripheral
blood lymphocytes and bone ,narrow were isolated (13) and analyzed for presence of virus. The spleens were first weighed before
homogenization. The tissues were sonicated in medium on ice,
and the presence of virus was determined by plaque assay (13). All
samples were stored at - 7 0 ~ before virus titration.
Infectious Center Assay. The frequency of persistently infected
lymphocytes was determined by assaying for infectious centers by
overlaying single-cell suspensions oil monolayers of NIH 3T3
cells. Various dilutions of the spleen and lymph node cells were
plated with 5 • 10~ NIH 3T3 cells, incubated overnight at 37~
and then overlaid with C M C / 2 • medium. The cells were cocultured for 5-6 d, the overlay was removed, and plaque counts
were determined as described above. Aliquots of 107 (or higher)
spleen or lymph node cells were also sonicated and assayed for the
presence of infectious virus.
Redirected Cytotoxicity Assay. Single-cell suspensions of lymph
nodes and spleen, or BAL cells that were first adsorbed on plastic
to remove most of the macrophages, were assayed for 6 h on
SlCr-labeled FcR + P815 ceils in the presence of the 2C11 mAb
to CD3e (26, 27). The level of specific StCr release is a measure
of the total cytotoxicity mediated by all activated T cells. For assays involving in vitro depletions, cell populations were incubated
with the appropriate mAb for 30 min at 4~ then the cells were
washed and incubated for 45 rain at 37~ in a cocktail of rabbit
(Accurate Chemical and Scientific Corp., Westbury, NY) and
guinea pig complement (Cedar Lane Laboratories, Hornby, Ontario).
Lymphocyte Depletion and Flow Cytometry. Mice in some experiments were depleted of CD4 + or CD8 + T cells by in vivo
treatment with the GK1.5 or 2.43.1 mAbs, respectively, which
were administered for at least 8 d at 2-3-d intervals (25, 28). The
prevalence of the various lymphocyte subsets was detemfined (29)
in two-color mode on a FACScan| (Becton Dickinson & Co.,
Mountain View, CA) with Lysis II software. The staining reagents were 53.6.7-FITC anti-CD8c~, H129.19-PE anti-CD4 §
and 1KA3-682 FITC anti-B220, all of which were purchased
from Pharmingen (San Diego, CA) and are not blocked by the
mAbs used for the in vivo depletions.
Cytokine Analysis. The single-cell ELISPOT assay was used
to quantify cytokine producing cells in freshly isolated BAL populations (30). Spleen and lymph node cells were restinmlated in
vitro with MHV-68-infected stimulator cells (31), and culture
supernatants were assayed by ELISA for the presence of IFN-%
IL-2, IL-4, IL-5, IL-6, and IL-10, as described previously (32).
The results are expressed as the percentage of spot-forming cells
(ELISPOT), or as nanograms (IL-10), or units per ml of culture
supernatant (ELISA).
Results
Virus Distribution during the Primary Stage of Infection.
The kinetics and peak titers of the initial virus growth
phase in the respiratory tract after i.n. inoculation with
M H V - 6 8 (4 X 103 PFU) were comparable for the M H C
class I1 + / + and - / mice (Fig. I a). Lower titers of
M H V - 6 8 were also detected in the adrenals on days 3 and
6 (Fig. 1 b), and in the thymus on day 6 (data not shown),
indicating that the virus had disseminated rapidly via the
blood in both groups of mice. Even so, the spectrum of organs supporting productive infection detectable by plaque
assay appears to be very limited. M H V - 6 8 could not be re-
LethalActivation of a ",/-Herpesvirus
a
9 +/+
6
Lung
r-I-I-
5
4
3
~
1
0
~
2o- 25
3
b
I>
,' 8o859o
13o 8
Adrenal
gland
2
O =
= i
o
5
,o
I
15
i
2o-25
;;
I
I I k
/p
728o85; "13o135
Days After Infection
Figure 1. MHV-68 replication in M H C class II + / + and - / - mice:
infectious virus tilers are shown for the lungs (a) and the adrenal glands (b)
of the + / + ( I ) and - / - mice ([]). Virus tilers are depicted as the mean
loglo P F U / m l of a 10% tissue sonicate, (+- SD), for three to five mice per
timepoint for three to tour separate experiments. Results for single experiments at 16, 17, 76, 132, and 133 d after infection are also included. The
limit of detection for the lung was 1 PFU, and 5 PFU for the adrenal
gland.
10000
a
]
i
Spleen
10000
b
63
O
O
rGL
10
10
5"
1
1
E
0.1
0.1
1 3
10000
1000
d 9§
100
10
1
10
1
0.1
0.1
3
10 12 13 15 16 17 2230
70 76 90
132133
3 6 1012131516172230
DaysAfterInfection
865
Cardinet al.
132 133
BM
IN-/-
100
o
r
i1V"I/1
11iI'
1 3 6 10 13 15 16 17 22 30//////70 76 90
132133
111i
1000
co
O
707690
CLN
r
O
1012131516172230
covered during this acute phase from tissue sonicates of the
pancreas, salivary glands, kidney, or liver, (data not shown).
Replication in the liver and kidneys has been reported for
BALB/c mice (17).
This virus does, however, quickly establish a latent infection in large numbers orB lymphocytes (19). Cocultivation
with permissive NIH 3T3 fibroblasts established the presence of substantial numbers of cells carrying MHV-68 in
the cervical lymph nodes (CLN) from day 3, and in the
mediastinal lymph nodes (MLN) and spleen from day 6
(Fig 2, a-c). Infectious centers were also detected subsequent to day 15 in the bone marrow (BM, Fig. 2 d), peripheral blood cells, and the mesenteric, axial, and brachial
lymph nodes (data not shown). The lymph nodes, spleen,
bone marrow, and blood cells yielded very few (or no)
plaques after sonication, indicating that MHV-68 is predominantly in a nonreplicating (or latent) status within the
lymphoid compartment.
Virus liters were greatly diminished by day 10 or 13 after
infection for lung sonicates from the + / + and - / - mice,
respectively, with both being negative by day 15 (Fig. 1 a).
Similarly, the adrenal samples were all negative by day 10
(Fig. 1 b). Despite the termination of the replicative phase,
however, there was no concurrent decrease in the frequencies of latently infected B cells (17) in the lymphoid tissue
(Fig. 2). The numbers ofMHV-68 + cells in the spleens of
the - / - mice (Fig. 2 a) were, however, consistently lower
through day 15, reflecting that the extent of splenomegaly
is generally much less in the absence of the CD4 + subset
(Fig. 3). In general, this analysis of the acute phase of
MHV-68 infection in MHC class II - / - B6 (H-2 b) mice
confirms and extends the conclusions reached from earlier
experiments with CD4-depleted BALB/c (H-2 a) mice (19,
tI
707690
132133
Figure 2. Profiles of MHV-68 latency are shown for lymphoid tissues
from the + / + and - / mice.
Lymphocytes from the (a) spleens;
(b) MLN; (c), CLN; and (d) BM o f
+ / + ( I ) and - / - ([~) mice were
isolated at different times after infection for infectious center assay. The
results are depicted at the mean
number (+ SD) of infectious centers
per 107 lymphocytes pooled from
three to five mice per timepoint for
repeat or single experiments (see legend to Fig. 1). Timepoints where no
infectious centers were detected are
indicated by asterisks. Otherwise,
timepoints with no data indicate that
the samples were not assayed.
0.5
9 §247
k~-/0.4
[ ] +/+ UN
E
[]
0.3
v
*/* MK
[ ] -/- UN
[]
e-
-/- MK
0.2
l..
0.1
0.O
1
3
6
10
13
15
16
17
30
22
70
76
Days After Infection
26). An important addition to these studies is that all the
lymph nodes, the bone marrow, and peripheral blood cells
harbor latent virus, indicating that these persistently infected cells are widely disseminated in the infected mouse.
Characteristics of the Acute Host Response. T h e fall in l u n g
M H V - 6 8 titers (Fig. 1 a) was a c c o m p a n i e d by a dramatic
increase (Fig. 4, c and d) in the m a g n i t u d e o f the cell p o p u lation recovered by BAL. T h e inflammatory cells were
generally m o r e than 30% m o n o c y t e / m a c r o p h a g e s , with the
r e m a i n i n g l y m p h o c y t e p o p u l a t i o n b e i n g d o m i n a t e d by the
C D 8 + subset (Fig. 4, a and b). In the + / + mice, the l o w e r
n u m b e r s o f C D 4 + T cells and few B220 + B ceils are c o n sistent with the pattern f o u n d for other virus infections o f
the respiratory tract (25, 28, 33). As yet, there is n o repro-
+/+
90
132 133
Figure 3. The extent of splenomegaty was determined by
weighing spleens from + / +
(I) and - / - ([~) nfice at different timepoints after MHV-68
infection. The spleens were into
preweighed tubes, and the results
are expressed as mean (+ SI))
spleen weight (grams) for groups
of three to five mice per timepoint. Spleens from uninfected
(UN) and mock-infected (MK)
nfice were measured for control
weights, but were not included
for each timepoint.
ducible virus-specific CTL assay developed for MHV-68.
Measuring the cytotoxic activity of all CD3e + T cells by
the redirected C T L assay (26, 27), h o w e v e r , s h o w e d the
e m e r g e n c e o f p o t e n t effectors in the BAL by day 10 (Fig. 5
a). T h e levels o f lysis were higher for the M L N and spleen
(Figs. 5, b and c), which, t h o u g h they are n o t supporting virus replication (Fig. 1), they do c o n t a i n large n u m b e r s o f
latently infected B lymphocytes (Fig. 2). A n t i b o d y depletion experiments with the + / + mice showed that the m a jority o f this C T L activity is mediated b y C D 8 + T cells, b u t
the C D 4 + T cells m a y also be m a k i n g some c o n t r i b u t i o n
(Fig. 5 d and Table 1).
Previous experiments with M H V - 6 8 in + / + mice have
s h o w n that the cytokine response to this virus is d o m i n a t e d
-/-
80 / a
I CD8
so I
[] s22o
co4
[]
8O
b
60
40
~. 2o
20
0
BAL
A
X
0
1.6
1.4
1.2
1.0
1.6
1.4
1.2
1.0
9
E= ,e
z
0
,4
.2
II,,,,
,tl,
, qilt
0 6 10 13 15 30 ## 7 0 9 0 / ' 1 3 3
6
d
/
.8
.6
.4
.2
0
0 6 10 13 15 3
Days After Infection
866
Lethal Activation ofa y-Herpesvirus
Figure 4. The characteristics of the inflammatory response in the lung are shown for the
MHV-68-infected + / + and - / - mice. The
BAL population was collected and pooled
from three to five mice per timepoint ill single or repeat experiments (see to Fig. 1). After
absorbance on plastic dishes, cell numbers
were determined (c and d)> and the samples
were analyzed by flow cytomet~" (,i and b).
70
Figure 5. Effector CTL activity from the + / + ( m - ) and - / 40 F
(~3) mice was determined as spen (+1*)
50
[]
cific SlCr release (6-h assay) for
r[] (q-)
freshly isolated BAL, MLN, and
3O ~40
spleen populations (at-, respec[]
30
tively) incubated with FcR_+
20 F
[]
P815 targets binding the 2 C l l
[]
o)
20
mAb to CD3-e (redirected ass
10 F
"3" 10
9
i
say), at an E/T of 40:1. TimeO
f[]
points through 70 d after infec,
/
/
,
,
,I,//,
,//_L
I I I I
I
I
I
i
iiii
I
OI D i
0-" i C
tion are from two separate
5
10
15 2 0 25 3 0 70 80 90 130135
10
15 2 0 2 5 3 0 70
0
5
80 90 "[30135
Q..
experiments with three to five
09
50
70
mice per group, the day 90 reC
9
SPL
sults are for a single experiment
(1)
d
[ ] Non-depleted
60
O
40
with three to five mice per
m CD4[]
50
group, while the day 133 results
n
[ ] CDOrepresent separate assays of three
30
T
+/+
40
~
9 9
mice per group. (d) Freshly iso30
lated BAL and spleen popula20
[]
tions were depleted of CD4 +
[]
n
20
(m) or CD8 + (V1) lymphocytes
10
10
by in vitro treatment with the
GK1.5 or 2.43,1 mAbs, respeci//i
I
I
I
I
I
I//l
I
0
tively, followed by incubation
5
10
15 ~'0 25 3 0 70 80 90 130135
all0 Spl
d60 Spl
610 BAL
with a cocktail of rabbit and
guinea pig complement. FracDays After Infection
tions of the cells were either untreated (nondepleted)or treated with complement alone (mock). Results represent the means (+ SD) of three separate experiments for nondepleted, C1)4depleted, and CDS-depleted samples, The mock treatment results are from two separate experiments.
50
a
60
D []
-b
BAL
[]
MLN
i
IB
Table
1.
[]D
[]
T Cell Depletion of Persistently Infected Mice
Percent o f
specific
SlCr release
Infectious centers$
Cytokines
Treatment
Virus titer in lung
(log10 PF U / m l )
Spleen
MLN
CLN
BAL
Spleen
IL-6
-/-
Saline
0.98 + 0.30
70
194
39
35.0
7.4
9.6
Day 84
CD4
1.43 + 0,58
60
88
40
ND
18.0
16.2
2.3
CD8-
3.66 _+ 0,58*
170
191
442
ND
7.3
15.3
3.2
-/-
Saline
2.2 + 0.04
130
(12.4 _ 9.75)
43.4
7.7
12.4 + 16.1
0
Day 114
CD8-
3.7 + 1.2"
150
(51.2 + 47.8)
ND
0
30.9 • 21.5
0
31.5 +_ 37.1
Group
IFN-y
U/ml
0
+/+
Saline
0
4
(0)*
44.5
32.9
9.6 + 21.5
Day 80
CD4
0
6
(0)
38.4
26.9
22.6 + 4.7*
8.1 +_ 6.8
CD8-
0
52
17.6
10.1
29.7 • 12.7"
83.5 + 55.0*
(1.6 + 0.8)*
The mice were treated with the GK1.5 mAb to CD4, the 2.43.1 mAb to CD8, or with saline at 2-3-d intervals commencing 10 d before sampling.
The percent o f CD8 + T ceils in the spleens o f the CD8-depleted mice were 0.3 (day 84), 0.5 (day 114), and 1.3 (day 80)_ The CD3e-dependent
CTL assay (26, 27) is described in the legend to Fig. 5. Cytokines (mean _+ SD) were measured by ELISA o f cu]ture supernatants from restimulated
spleen cells as described in the legend to Fig. 6.
*Significantly different (P <0.05 or <) from the saline control by Wilcoxon rank analysis (virus data) or, for the cytokine results, Mann U. Whitney
rank sum, test or Student's t test, depending on whether the data was nomlally distributed.
*The day 84 results show infectious centers per 10 v cells from pooled spleen, MLN, or CLN populations (five mice per group). The results at the
other timepoints are for pooled (per 107 cells) or individual (per 10f' cells, mean -+ SD, in parentheses) spleen cells. At day 114, - / - results are for
five mice (+ saline) and four mice (CD8-), one o f which was severely debilitated and was harvested on the previous day. At day 80, the + / + results
are for six mice (+ saline) and five mice (CDS-).
ND, not done because ofinsu~cient cell numbers.
867
Cardin et al.
b
a
100
4.5[ I
4.0 IFN-7
90
80
3.5
70
cD
o'-"l
)
60
r
O
50
40
30
3.0
E
o
2.5
LL
09
2.0
O
1.5
20
1.0
10
0.5
0
0
15
30
15
Days after Infection
30
by C D 4 + T cells producing IFN-'y and several cell types
(including acutely infected B lymphocytes) making IL-6
(reference 31 and unpublished data). This pattern was confirmed in the present study, with the amounts o f IFN-~/
that could be detected after in vitro restimulation of spleen
cells from the - / - mice being much lower, while the levels o f IL-6 were comparable for the two groups (Fig. 6 a).
In contrast to the results for the spleen, the numbers o f
IFN-',/-producing cells detected by single-cell E L I S P O T
analysis o f the BAL were similar for the + / + and - / mice (Fig. 6 b). Activated C D 8 § T cells are generally considered to produce I F N - y , but at a lower level than the
C D 4 + subset. If this E L I S P O T analysis is assaying virusspecific effectors, the prevalence in the BAL at the acute
phase of M H V - 6 8 infection is in the range of 1:20 for the
T cell component in both groups o f mice (compare Figs. 4
and 6 b). Analyzing sorted CD8 + T cells from the BAL has
not been successful (data not shown), perhaps because these
highly activated lymphocytes require support from m o n o cyte/macrophages for survival during the 24-h incubation
time of the E L I S P O T assay (30).
Virus Reactivation and Persistence. Virus was again detected consistently in lungs and adrenals from the - / mice subsequent to day 22 after the initial challenge, while
the only evidence o f virus replication in the + / + group
was a single plaque in a lung sample from day 90 (Fig. 1, a
and b). Virus was also detected in the kidneys o f one - / mouse at day 70, even though there was no evidence o f virus replication in this site during the acute infection (data
not shown). Furthermore, the numbers o f latently infected
cells in the lymph nodes and spleen (and peripheral blood
cells, but not the BM) decreased progressively for the + / +
mice, while the counts stayed high for the - / group
(Fig. 2). T h o u g h the lung titers in the - / - group were al868
15
30
Days after Infection
Figure 6. The cytokine production profiles are shown for
MHV-68-infected + / + and
- / - mice. Freshly isolated splenocytes from infected + / + (I)
and - / - ([]) mice were cultured with irradiated, MHV-68infected (MOI = (}.01) splenocytes isolated from uninfected
mice as APCs. The lymphocyte
cultures were incubated at 37~
and supernatants were collected
at 24, 72, and 96 h after infection
for detection of cytokine production by ELISA(31). (a) IFN-y
and IL-6 were measured in culture supernatants by ELISA. Resuits are expressed as the mean
units of IFN-'y or IL-6 per nil supematant after 72 h of stimulation. Little or no IL-2, IL-4, or
IL-5 was detected. (b) Frequencies of IFN-y-producing cells in
BAL populations were determined using the ELISPOT technique (30).
ways at least 10-fold lower than in the acute phase o f the
disease, these mice developed evidence o f wasting and 21
o f 25 died by day 133: the results at the late timepoints in
Figs. 1-4 are for the few survivors. The + / + mice all remained clinically normal.
Both the numbers o f C D 8 + T cells in the BAL (Fig. 4, b
and d) and the levels o f CD3~- dependent C T L activity
(Fig. 5 a) were much higher for the - / mice at the day
133 timepoint. This divergence from the + / + controls
was not seen for the spleen (Fig. 5 c), which contained significant populations o f latently infected B cells in both
groups (Fig. 2 a). In other virus models (such as influenza)
where the virus is rapidly eliminated, this CD3E-dependent
effector function does not persist beyond 2-3 wk after infection (Tripp, P,..A., unpublished data). T h o u g h the total
numbers o f C D 8 + T cells did not decline (data not shown),
restimulation o f the - / spleen cells in vitro after day 30
gave no indication of a significant I F N - y recall response
(Table 1). Spleen cells from + / + mice, however, continued to produce significant levels of IFN-',/ (data not
shown), as previously reported (31).
Maintenance then Loss of C D 8 + T Cell-mediated Control.
Given that the acute phase o f M H V - 6 8 infection is terminated by the C D 8 + subset, the obvious question was
whether the reactivation or reappearance of infectious virus
in the lungs and adrenals by day 22 in the - / - mice (Fig. 1)
reflected that the C D 8 + effectors rapidly become nonfunctional in the absence o f C D 4 + T cell "help." There is a
precedent for thinking that such "immune exhaustion"
(34, 35) can occur from experiments with lymphocytic
choriomeningitis virus (LCMV).
Depleting the - / - mice with the 2.43.1 mAb to C D 8 +
commencing at 74 or 104 d after infection established that
there is still some CD8 + T cell-mediated control of M H V -
Lethal Activation ofa y-Herpesvirus
68 at this late stage o f the disease process. In the first experiment (day 84, Table 1), removing the C D 8 + T cells
caused a > 4 0 - f o l d increase in lung virus titers within 10 d
o f commencing the mAb treatment, compared with a > 3 fold effect in - / - mice given the GK1.5 mAb to C D 4 + as
a control. The repeat experiment showed a > 3 0 - f o l d increase after in vivo elimination o f the C D 8 + subset in the
-/mice (day 114, Table 1), while no virus was detected
in lung sonicates from + / + controls that were treated concurrently (Table 1). Only the CDS-depleted - / - animals
showed any signs o f obvious debility and, in the second experiment, the mice were weighed every 2 d to confirm the
impression o f wasting. All - / - mice exhibited weight loss
after treatment with the 2.43.1 mAb, with one decreasing
40% within 6 d. This mouse was sampled 1 d early, and had
the highest lung virus titer yet recorded (105) during the
phase o f M H V - 6 8 reactivation (data not shown).
The numbers oflatently infected B cells in - / - pooled
spleen populations also tended to increase after the removal
o f the C D 8 + subset (day 84 and 114, Table 1), with this
treatment being statistically significant for the + / + mice
that were analyzed on day 80 (Table 1). The + / + mice
also showed evidence of reduced CD3~-dependent C T L
activity in the BAL and spleen after both C D 4 + and C D 8 +
T cell depletion, but only the latter treatment had any effect on the frequency o f M H V - 6 8 + B cells, which were still
much less prevalent compared to the - / - mice (Table 1). It
thus seems that the numbers o f persistently infected B ceils
are being continuously controlled by C D 8 +, but not
C D 4 +, effectors. However, this C D 8 + T cell surveillance
function becomes progressively compromised in the longterm absence o f the C D 4 + subset.
Discussion
N o comparable generalized recrudescence o f a D N A virus infection has been described for any rodent model. Depleting C D 4 + T cells with an appropriate mAb led to defective control o f murine cytomegalovirus (CMV) in the
salivary gland, and to reactivation after such treatment o f
latently infected mice (36, 37, 13). Unlike M H V - 6 8 (19),
systemic C M V infection can be limited by C D 4 + T cells
acting in the absence o f the C D 8 + subset (38). The effector
mechanism is I F N - y dependent, with C M V dissemination
during recurrent infection being facilitated by subversion o f
the specific antibody response. Failure to neutralize M H V 68 could be contributing to the progressive involvement o f
new B lymphocyte and epithelial cell targets in the M H V 68-infected M H C class II - / mice. However, the consequences o f eliminating the CD8 + subset late in the course
o f M H V - 6 8 infection are such that, even if the lack o f antibody is allowing indolent virus to spread, the extent o f the
infectious process is still being limited by the C D 8 + effector population for some months.
It is k n o w n that concurrent C D 4 + T help can sometimes, though not always, have a significant effect on the
acute clonal expansion and differentiation o f the virus-specific C D 8 + population (33). The CD8 + T cell response af869
Cardin et al.
ter infection with Herpes simplex virus was substantially diminished by mAb depletion o f the C D 4 + subset
commencing before virus priming (39). The - / mice
used here developed fewer virus-specific C D 8 + C T L precursors after infection with an influenza A virus (24),
though clearance from the lung was not compromised (24,
40), and the acute C T L response to L C M V and to Sendal
virus was substantially normal (41, 42). Results from the influenza experiments suggests that the smaller numbers o f
C T L precursors were transiently consumed to give a sufficient population o f C T L effectors (24). Comparable cytokine profiles were found for - / mice infected acutely
with Sendal virus or influenza virus, though the level o f
I F N - y production was much higher for the Sendal model
(24, 42).
The requirement for the C D 4 + subset to sustain effective
CD8 + T cell surveillance in the M H V - 6 8 model is likely
to reflect that the "helpers" function continuously to provide a cytokine-rich milieu in responding lymphoid tissue.
P, estimulation o f lymphocyte populations from M H V - 6 8
infected + / + mice has shown the long-term presence o f
I F N - y - p r o d u c i n g T cells (31). Little IL-2 is detected, and
there is no tendency for IL-4 producers to emerge in the
long-term in either the + / + or - / mice (data not
shown). The cytokines may be promoting continued clonal
expansion o f the CD8 + responder population, or diminishing the extent o f effector T cell loss by (for example)
blocking apoptosis. The in vivo dissection o f rates o f virusspecific T cell production and loss presents major difficulties with even the well-established viral, models (43, 44),
and is clearly not feasible at this early stage o f the analysis
with M H V - 6 8 .
Like M H V - 6 8 (19), L C M V cannot be eliminated by
C D 4 + T cells acting in the absence o f the C D 8 + subset
(45, 46). Mice infected with macrophage or lymphotropic
variants of L C M V develop a persistent infection, which is
eventually cleared in normal mice, but not in C D 4 depleted mice (35). The progressive exhaustion o f the
CD8 + subset in this and in the "high virus dose immune
paralysis" model o f L C M V infection (34) has obvious parallels to the events that we describe here in the M H C class
II - / mice infected with M H V - 6 8 . The differences are
that L C M V is k n o w n to infect a wide variety o f cell types
in vivo, (including a percentage o f t lymphocytes), but unlike the herpesviruses, this 1LNA virus has no mechanism
for establishing latent infection.
It is not clear whether the M H V - 6 8 replicative phase in
the lung and the latently infected B cells in lymphoid tissue
are controlled by the same or different sets o f virus-specific
effectors (47), and will not be understood until at least
some o f the epitopes recognized by the responding T cells
are defined. The mAb depletion experiments with B A L B / c
mice (19) had already established that the termination o f
the acute, replicative phase in epithelial sites is mediated by
the C D 8 + subset. Even so, the larger numbers o f M H V 68 + B cells in the persistently infected - / - mice may reflect that M H C class II-restricted C D 4 + effectors operate
to limit the extent of infection in the normal + / + animal.
However, treating the persistently-infected + / + mice
with an m A b to the C D 8 + but, not the C D 4 +, subset increased the numbers o f M H V - 6 8 + B cells. T h o u g h C D 4 +
C T L can be shown to develop after the infection o f mice
that lack M H C class I glycoproteins with (for example)
L C M V and Sendai virus (48, 49), there is currently no evidence that such effectors are functioning directly to eliminate latently infected B cells in the M H V - 6 8 model.
Infection o f M H C class II - / mice with M H V - 6 8
thus offers a unique experimental system for assessing the
mechanisms that underlie compromised i m m u n e surveillance in a model o f viral latency, and for screening possible
chemotherapy-, cytokine-, or cell-based protocols to limit
yHV-associated disease. This is the first time that we have
had the opportunity to analyze experimentally a situation
where there is continuing control o f a D N A virus by
C D 8 + effectors. The fact that the progressive escape from
effective surveillance by C D 8 + T cells is a direct consequence o f the absence o f the C D 4 + subset offers a direct
parallel to the situation in AIDS.
We thank Dr. A.A. Nash ibr sending us the MHV-68; I)rs. A.A. Nash, J. Sixbey, and R.A. Tripp ~br advice;
Jim Houston, Roseann Lambert, Mahnaz Paktinat, Anthony McMickle, and Mehdi Mehrpooya for technical assistance; and Vicki Henderson for typing the manuscript.
This study was supported by National Institutes of Health (;rants CA09346 and CA21765, and by the
American Syrian Lebanese Associated Charities (ALSAC).
Address correspondence to Dr. Peter Doherty, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105.
Receivedfor publication 9 April 1996 and in revisedform 19June 1996.
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